This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heparins consist of linear sulfated polysaccharides of that are polydisperse with respect to chain length, extent of sulfation, acetylation, and uronic acid epimerization. The most abundant repeating unit is (IdoA2S-GlcNS6S). A significant fraction of GlcN residues exist in acetylated form, and other modifications (free glucosamine) may be present at low levels. Low molecular weight heparins (LMWHs) have an average mass of 5000, and consist of a distribution of oligosaccharide chain lengths. They are produced by partial depolymerization of intact heparin, and the methods used differ among the various pharmaceutical LMWH products. A given chain length consists of a distribution of isomeric forms with respect to sulfation, acetylation, and epimerization. It is not possible to chromatographically purify individual isomers for LMWH chains. Therefore, LMWH preparations do not have a sequence in the sense applied to homogeneous biopolymers. It is more useful to assess the distribution of polysaccharide compositions by measuring their masses and abundances. These compositions will provide a useful measurement of the sameness of LMWH preparations. Given the recent problems with the heparin production system, the development of analytics for pharmaceutical preparations is of high priority. Towards these ends, we are developing several approaches for heparin analysis. First, heparins or LMWH are exhaustively depolymerized using enzymes and the resistant oligosaccharides analyzed using on- and off-line LC/MS. Tandem MS is used to characterize the glycoforms present in the resistant oligosaccharides. Second, a distribution of oligosaccharides is generated by partial depolymerization of heparin products and the glycan profiles determined using LC/MS with appropriate informatics approaches. Third, heparins and LMWH are analyzed directly using LC/MS.